The effects of inactivated SARS‐CoV‐2 vaccination and subsequent infection of pregnant mice on the behaviors of offspring

Abstract The mass inoculation of severe acute respiratory syndrome coronavirus 2 (SARS‐CoV‐2) vaccines to induce herd immunity is one of the most effective measures we can deploy in the fight against coronavirus disease 2019 (COVID‐19). Pregnant women are prone to a higher risk of COVID‐19, and maternal infection is a risk factor for a range of neurological disorders leading to abnormal behavior in adulthood. However, there are limited clinical data to support whether vaccination or infection post‐immunization in pregnant women can affect the behavioral cognition of fetuses in adulthood. In this study, human angiotensin‐converting enzyme 2 pregnant mice (F0 generation) were immunized with CoronaVac and then infected with SARS‐CoV‐2. Subsequently, we analyzed the behavioral cognition of their adult offspring (F1 generation) using the open‐field test and Morris water maze test. The adult F1 generation did not exhibit any impairments in spontaneous locomotor activity or spatial reference memory.


| INTRODUC TI ON
The pandemic of pneumonia caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has caused great pressure and resulted in tragic consequences to the global public health and medical system. A massive Coronavirus Disease 2019 (COVID- 19) vaccination campaign is underway worldwide. 1 Although pregnant women are prone to a higher risk of COVID-19, clinical trials for the vaccines excluded pregnant and lactating women. Maternal infection may not only lead to premature delivery, abortion, and other adverse pregnancy outcomes but also increase the possibility of infection after childbirth. 2,3 Epidemiological studies have found that prenatal infection is a risk factor for a range of neurological disorders, accompanied by an increased risk of schizophrenia, working memory defects, and executive dysfunction. 4 Maternal infection disrupts the immune balance between the maternal and fetal environments, leading to changes in the immune profile in the developing brain. 5 The infection of pregnant women leads to the increase in cytokines such as interleukin-6, which can pass through the placenta and act on placental cells to stimulate the production of downstream immune mediators in the uterus, and has become one of the risk factors for neurodevelopmental disorders in offspring. 6 However, very limited data regarding the safety and validity of the vaccine during pregnancy are available. Consequently, women who are planning to become pregnant feel uneasy about being vaccinated. Our previous research results showed that the serum neutralizing antibody (NAb) and S1-specific IgG titers of human angiotensin-converting enzyme 2 (hACE2) mice inoculated with CoronaVac (an inactivated SARS-CoV-2 vaccine) were 256-512 and 6400-25 600 arbitrary units (AU) at 35 days after immunization. 7,8 The NAb titers of offspring in the vaccination group and the vaccination/infection group were 64-256 and 8-32 AU at weaning day, and the S1-specific IgG titers were 1600-6400 and 800-3200 AU, indicating that the offspring successfully obtained NAb and IgG from the parental generation. 7 In addition, there was no abnormality in the number, sex ratio, growth, and development of offspring in each group. 7 In this research, hACE2 mice (F0 generation) were selected as a model to study the effects of immunization and post-immunization infection in F0 generation on adult offspring (F1 generation) behaviors. According to the general principles for technical review of preclinical safety evaluation of preventive biological products in China, the inoculation dose of vaccine, in principle, should achieve the best immune response in animals.
F0 generations were inoculated with a 100μl dose of CoronaVac at 0 and 28 days and infected with SARS-CoV-2 at 36 days. 9 Then, we investigated the potential effects on spontaneous locomotor activity and spatial reference memory of the offspring. We intended to provide basic research data to support the clinical inoculation of pregnant women and women of childbearing age with inactivated SARS-CoV-2 vaccine.  Figure 1A). The temperature was 23-25°C, and relative humidity was 55% ± 10%, with a 12-h light-dark cycle.

| Animal grouping and processing methods
The F1 generations were transferred to the laboratory to adapt to the environment 30 min before the start of each behavioral experiment. All animal procedures were approved by ILAS, PUMC (reference: GH21001).

| Morris water maze test
The Morris water maze consisted of a cylinder without a lid (diameter, 120 cm; height, 40 cm); the water level was 25 cm; a platform was located 1.5 cm below the water. Then, a white odorless substance was added to the water maze and stirred evenly. Next, the pool was divided into four quadrants, the platform was placed in the northwest zone, and the water temperature was maintained at 22-25°C. A nontransparent curtain was labeled with markers, and data were recorded and analyzed with a Nolduc Etho Vision XT9 image acquisition and analysis system. Experiments were conducted over a 6-day cycle; placement navigation was tested thrice a day between days 1 and 5. As the center of the quadrant arc was set as the delivery point, the F1 generations were placed randomly in the maze without repetition ( Table 1). The experimental procedure lasted for 1 min. If the F1 generations could stand on the platform for 5 s, then platform-seeking ability was considered to be successful; the time from water entry to successful platform localization was recorded as the latency. If the F1 generations were unable to stand on the platform for 1 min, then the latency would be recorded as 1 min. Cumulative movement distance was regarded as the latent movement distance. All of the mice were required to stand on the platform for 5 s regardless of whether they successfully sought out the platform. Space exploration experiments were carried out on day 6; the procedure lasted for 1 min, during which the platform was removed, and the mice were released from the southeast zone.

| Statistical analysis of data
Data analysis was carried out using GraphPad Prism 8.0 software.
One-way analysis of variance unpaired Student's t-test was used to compare three groups. *p < 0.05 indicated statistically significant difference.

| Inoculated with the inactivated SARS-CoV-2 vaccine and subsequent infection of F0 generation, the F1 generation exhibited no side effects on spontaneous locomotor activity
The open-field test results revealed that the differences in cumula-

| Inoculated with the inactivated SARS-CoV-2 vaccine and subsequent infection of F0 generation, the F1 generation showed no adverse effects on the spatial reference memory
According to data relating to place navigation in the Morris water maze, there were no significant differences in the latent movement

| DISCUSS ION
Maternal exposure to microbial infection or immune activation during pregnancy has a long-term impact on the development of the fetus after birth. Early research data show that the generation born during the pandemic of infectious diseases is at developmental risk. 10 The infection of pregnant women with viruses, such as rubella, respiratory tract infection, influenza, and bacterial infection, is a risk factor for numerous neurological and psychiatric disorders in their offspring. [11][12][13][14] For a subset of women, maternal infection may be an inducement, leading to an altered trajectory of fetal brain development. Neuroimaging and behavioral studies have provided further evidence that schizophrenic patients exposed to infection in utero exhibited a characteristic pattern of brain volume changes, accompanied by an increased risk of schizophrenia, working memory defects, and executive dysfunction. 6,15 Furthermore, the dramatic increase in inflammatory cytokine levels in the fetus may have adverse effects on neurodevelopment. 15,16 Animal studies have shown that activation of the maternal immune system by viral infection (e.g., the influenza virus) may alter the levels of cytokines in the placenta, amniotic fluid, and fetal brain, 17 thus affecting brain development and potentially inducing brain injury in the fetus. Pregnant rats were stimulated with immune activators, such as lipopolysaccharide, to produce strong immune responses, and the offspring exhibited similar changes in the brain and behavioral phenotypes with human disorders 18 ; these events may eventually result in executive dysfunction as well as spatial learning and memory impairment.
Pregnancy vaccination can provide not only direct protection against infectious diseases, and its side effects, but also passive immunity for the newborn. 19 Since December 2020, more than 185 000 pregnant women have been injected with COVID-19 mRNA vaccine, and clinical data showed no increased risks of any adverse obstetric outcome, including preterm birth, stillbirth, neonatal death, and congenital malformations. 20 In the global clinical trial of ChAdOx1 nCoV-19, the pregnancy outcome analysis of 50 women who were pregnant after vaccination showed no effect on fertility rates and pregnancy outcomes by vaccination before pregnancy. 21 The investigation of CoronaVac in pregnant women showed that two doses were 41% effective against symptomatic COVID-19 and 85% effective against severe COVID-19. 22  which does not require animals to be trained up front or restrict food and drinking water, 23 is particularly used to assess the spatial learning and memory of animals and can be very useful for investigating the effects of drugs in rodents. 24,25 In this study, hACE2 pregnant mice were inoculated with the inactivated SARS-CoV-2 vaccine and infected with SARS-CoV-2 after immunization. We

ACK N OWLED G M ENT
This work was supported by the National Key Research and Development Project of China (grant number: 2020YFA0707600).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflicts of interest.

E TH I C S A PPROVA L
All animal procedures were approved by the Institutional Animal Care and Use Committee at ILAS, PUMC (Reference: GH21001).